Periodic Reporting for period 1 - SLYDIV (Unveiling the molecular coordination between the cell division machinery and S-layer biogenesis in Bacillus anthracis)
Reporting period: 2023-09-01 to 2025-08-31
Summary of the context and overall objectives of the project
The SLYDIV project was designed to address a fundamental question in microbiology: how do bacteria coordinate the assembly of their surface layers (S-layers) with cell growth and division? S-layers are proteinaceous crystalline arrays that coat the surface of many bacteria and archaea, playing critical roles in structural integrity, environmental resilience, and host-pathogen interactions. Despite their ubiquity, the molecular mechanisms governing S-layer biogenesis and their integration with the bacterial cell cycle remain poorly understood.
This knowledge gap is particularly relevant in the context of antimicrobial resistance and the need for novel therapeutic strategies. In pathogens such as Bacillus anthracis, the S-layer is essential for virulence and cell envelope stability, making it a promising target for antimicrobial development. Similarly, in industrially relevant species like Corynebacterium glutamicum, understanding S-layer dynamics offers opportunities for synthetic biology and bioengineering applications.
The original objectives of the SLYDIV project were to:
1. Determine the atomic structure of the S-layer protein in B. anthracis.
2. Investigate the spatial and temporal dynamics of S-layer assembly, particularly its coordination with polar growth.
3. Unveil the molecular coordination between the divisome and S-layer biogenesis
These objectives were pursued using a multidisciplinary approach combining cryo-electron microscopy, X-ray crystallography, microbiology, and live-cell imaging. The project has yielded significant advances, including the first complete atomic model of the B. anthracis S-layers Sap and EA1, the discovery of polar S-layer assembly in C. glutamicum, and the development of a modular S-layer engineering platform.
In addition to these planned outcomes, the project also led to an unexpected but highly valuable collaboration that expanded the scientific scope. In partnership with colleagues at the VIB-VUB Center for Structural Biology, I contributed to the discovery and characterization of a novel class of protein nanofibers, A-ENA (alpha-helical endospore appendages) produced by Bacillus thuringiensis. These fibers form a robust extracellular matrix that clusters spores and parasporal bodies (PSBs), enhancing the insecticidal efficacy of this biopesticide. This work, although not part of the original SLYDIV objectives, provided a unique opportunity to deepen my expertise in bacterial surface structures and their functional roles in pathogenesis and biotechnology.
The broader impact of the SLYDIV project lies in its contribution to EU policy priorities in health, biosecurity, and sustainable agriculture. By advancing our understanding of bacterial surface architecture and developing tools for its manipulation, the project supports the development of next-generation antimicrobials, bio-based production systems, and environmentally friendly pest control strategies. The integration of fundamental research with translational potential exemplifies the MSCA mission to foster scientific excellence and societal impact across Europe.
This knowledge gap is particularly relevant in the context of antimicrobial resistance and the need for novel therapeutic strategies. In pathogens such as Bacillus anthracis, the S-layer is essential for virulence and cell envelope stability, making it a promising target for antimicrobial development. Similarly, in industrially relevant species like Corynebacterium glutamicum, understanding S-layer dynamics offers opportunities for synthetic biology and bioengineering applications.
The original objectives of the SLYDIV project were to:
1. Determine the atomic structure of the S-layer protein in B. anthracis.
2. Investigate the spatial and temporal dynamics of S-layer assembly, particularly its coordination with polar growth.
3. Unveil the molecular coordination between the divisome and S-layer biogenesis
These objectives were pursued using a multidisciplinary approach combining cryo-electron microscopy, X-ray crystallography, microbiology, and live-cell imaging. The project has yielded significant advances, including the first complete atomic model of the B. anthracis S-layers Sap and EA1, the discovery of polar S-layer assembly in C. glutamicum, and the development of a modular S-layer engineering platform.
In addition to these planned outcomes, the project also led to an unexpected but highly valuable collaboration that expanded the scientific scope. In partnership with colleagues at the VIB-VUB Center for Structural Biology, I contributed to the discovery and characterization of a novel class of protein nanofibers, A-ENA (alpha-helical endospore appendages) produced by Bacillus thuringiensis. These fibers form a robust extracellular matrix that clusters spores and parasporal bodies (PSBs), enhancing the insecticidal efficacy of this biopesticide. This work, although not part of the original SLYDIV objectives, provided a unique opportunity to deepen my expertise in bacterial surface structures and their functional roles in pathogenesis and biotechnology.
The broader impact of the SLYDIV project lies in its contribution to EU policy priorities in health, biosecurity, and sustainable agriculture. By advancing our understanding of bacterial surface architecture and developing tools for its manipulation, the project supports the development of next-generation antimicrobials, bio-based production systems, and environmentally friendly pest control strategies. The integration of fundamental research with translational potential exemplifies the MSCA mission to foster scientific excellence and societal impact across Europe.
Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far
During my MSCA postdoctoral fellowship at the VIB-VUB Center for Structural Biology, I investigated the molecular architecture, assembly mechanisms, and physiological roles of bacterial surface layers (S-layers), with a focus on their coordination with cell division and envelope biogenesis. The project combined cryo-electron microscopy (cryo-EM), X-ray crystallography, microbiology, and live-cell imaging to address fundamental questions in bacterial cell envelope biology.
A major achievement was the high-resolution structural characterization of the Sap S-layer protein from Bacillus anthracis. Using cryo-ET, subtomogram averaging, X-ray crystallography, and molecular dynamics simulations, we solved the complete atomic model of the Sap lattice. This work revealed a conformational switch from a condensed monomeric state to an extended post-assembly protomer, enabling lattice formation. I identified four key interdomain interfaces that stabilize the lattice and demonstrated their functional relevance through mutagenesis and biophysical assays. These findings provide a mechanistic framework for understanding S-layer assembly and offer new targets for antimicrobial development.
In parallel, I led a study on the PS2 S-layer of Corynebacterium glutamicum, where I determined its structure at 2.5 Å resolution. I discovered that PS2 forms a semi-permeable, hexameric-trimeric lattice anchored into the mycomembrane via a coiled-coil transmembrane domain. Using a SpyTag/SpyCatcher system, I engineered the PS2 lattice for covalent surface display and demonstrated its functionality both in vitro and in vivo. Through pulse-chase fluorescence microscopy, I showed that PS2 assembly occurs exclusively at the cell poles, coordinated with the elongasome. This was the first demonstration of polar S-layer biogenesis and revealed a new model for S-layer growth in polar-growing bacteria.
In addition to the original project objectives, I contributed to a collaborative study that led to the discovery and structural characterization of a novel class of protein nanofibers, A-ENA (alpha-helical endospore appendages) produced by Bacillus thuringiensis. These fibers form a robust extracellular matrix that clusters spores and parasporal bodies (PSBs), enhancing the insecticidal efficacy of this biopesticide. I performed the biological and biochemical assays demonstrating that A-ENA acts as a virulence factor by promoting spore–toxin co-localization. I also showed that recombinant A-ENA fibers can be produced in E. coli and used to enhance the virulence of other Bt strains in a non-GMO manner. This work, although not part of the original SLYDIV objectives, significantly expanded my expertise in bacterial surface structures and their functional roles in pathogenesis and biotechnology.
Collectively, these studies resulted in multiple high-impact publications and structural data depositions, and they significantly advanced our understanding of S-layer biology, bacterial envelope dynamics, and the engineering of surface structures for translational applications.
A major achievement was the high-resolution structural characterization of the Sap S-layer protein from Bacillus anthracis. Using cryo-ET, subtomogram averaging, X-ray crystallography, and molecular dynamics simulations, we solved the complete atomic model of the Sap lattice. This work revealed a conformational switch from a condensed monomeric state to an extended post-assembly protomer, enabling lattice formation. I identified four key interdomain interfaces that stabilize the lattice and demonstrated their functional relevance through mutagenesis and biophysical assays. These findings provide a mechanistic framework for understanding S-layer assembly and offer new targets for antimicrobial development.
In parallel, I led a study on the PS2 S-layer of Corynebacterium glutamicum, where I determined its structure at 2.5 Å resolution. I discovered that PS2 forms a semi-permeable, hexameric-trimeric lattice anchored into the mycomembrane via a coiled-coil transmembrane domain. Using a SpyTag/SpyCatcher system, I engineered the PS2 lattice for covalent surface display and demonstrated its functionality both in vitro and in vivo. Through pulse-chase fluorescence microscopy, I showed that PS2 assembly occurs exclusively at the cell poles, coordinated with the elongasome. This was the first demonstration of polar S-layer biogenesis and revealed a new model for S-layer growth in polar-growing bacteria.
In addition to the original project objectives, I contributed to a collaborative study that led to the discovery and structural characterization of a novel class of protein nanofibers, A-ENA (alpha-helical endospore appendages) produced by Bacillus thuringiensis. These fibers form a robust extracellular matrix that clusters spores and parasporal bodies (PSBs), enhancing the insecticidal efficacy of this biopesticide. I performed the biological and biochemical assays demonstrating that A-ENA acts as a virulence factor by promoting spore–toxin co-localization. I also showed that recombinant A-ENA fibers can be produced in E. coli and used to enhance the virulence of other Bt strains in a non-GMO manner. This work, although not part of the original SLYDIV objectives, significantly expanded my expertise in bacterial surface structures and their functional roles in pathogenesis and biotechnology.
Collectively, these studies resulted in multiple high-impact publications and structural data depositions, and they significantly advanced our understanding of S-layer biology, bacterial envelope dynamics, and the engineering of surface structures for translational applications.
Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)
The SLYDIV project has delivered several scientific and technological advances that significantly extend the current state of the art in bacterial cell envelope biology, structural microbiology, and bioengineering.
A key achievement was the complete atomic-level characterization of the Sap S-layer protein from Bacillus anthracis, revealing a novel conformational switch and four stabilizing interdomain interfaces. These findings provide a mechanistic framework for S-layer assembly and offer new molecular targets for antimicrobial development, particularly relevant in the context of antimicrobial resistance and biosecurity.
In Corynebacterium glutamicum, I solved the PS2 S-layer structure at 2.5 Å resolution and discovered that its assembly is spatially restricted to the cell poles, coordinated with the elongasome. This was the first demonstration of polar S-layer biogenesis and challenges the prevailing model of mid-cell insertion. I also developed a modular S-layer engineering platform using SpyTag/SpyCatcher technology, enabling programmable surface display in a GRAS industrial host. This innovation opens new avenues for applications in vaccine delivery, biosensing, and biocatalysis.
In addition to the original project objectives, I contributed to a collaborative study that led to the discovery and structural characterization of a novel class of protein nanofibers, A-ENA (alpha-helical endospore appendages) produced by Bacillus thuringiensis. These fibers form a chemically and physically robust extracellular matrix that clusters spores and parasporal bodies (PSBs), enhancing the insecticidal efficacy of this biopesticide. I demonstrated that A-ENA acts as a virulence factor and that its recombinant production in E. coli enables non-GMO functionalization of other Bt strains. This work introduces a new bioengineering tool for improving the performance of biological pest control agents and contributes to sustainable agriculture and integrated pest management strategies.
These innovations have led to two patent applications:
• WO2025/046122: This patent covers the design, production, and application of A-ENA nanofibers as highly stable, self-assembling bionanomaterials. It includes methods for recombinant production and their use in modifying bacterial endospores and enhancing their activity or pathogenicity.
• EP25162107.4 (priority filing): This application focuses on the use of A-ENA nanofibers to enhance the pesticidal activity of Bt strains, offering a non-GMO strategy to improve the efficacy of biocontrol agents.
Potential Impacts and Future Needs
• Further Research: Continued investigation into the regulation, secretion, and host interactions of S-layers and A-ENA fibers will be essential to fully exploit their therapeutic and biotechnological potential.
• Demonstration and Scale-Up: The PS2 display platform and A-ENA-based biofilm engineering should be validated in industrial and agricultural settings to assess scalability, robustness, and regulatory compliance.
• Commercialisation and IPR: The A-ENA nanofiber technology is protected through patent filings. Further support from technology transfer offices will be needed to explore licensing opportunities and partnerships with biotech and agri-tech companies.
• Internationalisation: The project has already fostered international collaborations and is well-positioned to expand into global networks focused on antimicrobial innovation, synthetic biology, and sustainable agriculture.
• Regulatory and Standardisation Frameworks: For therapeutic and agricultural applications, early engagement with regulatory bodies will be crucial to define safety, efficacy, and environmental impact standards.
In summary, the SLYDIV project has not only fulfilled its original objectives but also generated high-impact, patentable innovations through interdisciplinary collaboration. These outcomes position the project at the forefront of microbial structural biology and synthetic biology, with clear pathways toward societal, environmental, and industrial impact.
A key achievement was the complete atomic-level characterization of the Sap S-layer protein from Bacillus anthracis, revealing a novel conformational switch and four stabilizing interdomain interfaces. These findings provide a mechanistic framework for S-layer assembly and offer new molecular targets for antimicrobial development, particularly relevant in the context of antimicrobial resistance and biosecurity.
In Corynebacterium glutamicum, I solved the PS2 S-layer structure at 2.5 Å resolution and discovered that its assembly is spatially restricted to the cell poles, coordinated with the elongasome. This was the first demonstration of polar S-layer biogenesis and challenges the prevailing model of mid-cell insertion. I also developed a modular S-layer engineering platform using SpyTag/SpyCatcher technology, enabling programmable surface display in a GRAS industrial host. This innovation opens new avenues for applications in vaccine delivery, biosensing, and biocatalysis.
In addition to the original project objectives, I contributed to a collaborative study that led to the discovery and structural characterization of a novel class of protein nanofibers, A-ENA (alpha-helical endospore appendages) produced by Bacillus thuringiensis. These fibers form a chemically and physically robust extracellular matrix that clusters spores and parasporal bodies (PSBs), enhancing the insecticidal efficacy of this biopesticide. I demonstrated that A-ENA acts as a virulence factor and that its recombinant production in E. coli enables non-GMO functionalization of other Bt strains. This work introduces a new bioengineering tool for improving the performance of biological pest control agents and contributes to sustainable agriculture and integrated pest management strategies.
These innovations have led to two patent applications:
• WO2025/046122: This patent covers the design, production, and application of A-ENA nanofibers as highly stable, self-assembling bionanomaterials. It includes methods for recombinant production and their use in modifying bacterial endospores and enhancing their activity or pathogenicity.
• EP25162107.4 (priority filing): This application focuses on the use of A-ENA nanofibers to enhance the pesticidal activity of Bt strains, offering a non-GMO strategy to improve the efficacy of biocontrol agents.
Potential Impacts and Future Needs
• Further Research: Continued investigation into the regulation, secretion, and host interactions of S-layers and A-ENA fibers will be essential to fully exploit their therapeutic and biotechnological potential.
• Demonstration and Scale-Up: The PS2 display platform and A-ENA-based biofilm engineering should be validated in industrial and agricultural settings to assess scalability, robustness, and regulatory compliance.
• Commercialisation and IPR: The A-ENA nanofiber technology is protected through patent filings. Further support from technology transfer offices will be needed to explore licensing opportunities and partnerships with biotech and agri-tech companies.
• Internationalisation: The project has already fostered international collaborations and is well-positioned to expand into global networks focused on antimicrobial innovation, synthetic biology, and sustainable agriculture.
• Regulatory and Standardisation Frameworks: For therapeutic and agricultural applications, early engagement with regulatory bodies will be crucial to define safety, efficacy, and environmental impact standards.
In summary, the SLYDIV project has not only fulfilled its original objectives but also generated high-impact, patentable innovations through interdisciplinary collaboration. These outcomes position the project at the forefront of microbial structural biology and synthetic biology, with clear pathways toward societal, environmental, and industrial impact.